Microbial Control of Host Intercellular Communication

项目总结/摘要 多细胞生物利用细胞间通讯来协调细胞功能和维持组织 体内平衡最近的研究表明，这种通信部分是由细胞器的交换驱动的（通过 反式内吞作用）和直接穿过细胞-细胞连接的机械信号。这种沟通的失调 导致癌症和心血管疾病。尽管它很重要，但我们缺乏一个基本的分子 由于细胞生物学工具的数量有限， 能够探测细胞间接触的分子机制。本建议旨在阐明 调节机制的途径，认为控制细胞间的沟通，通过研究他们是如何 在微生物控制下进行操作。单核细胞增生李斯特菌通过人类传播 细胞间扩散是一种囊泡介导的细胞间交换形式， 模仿宿主的反式内吞作用李斯特菌通过调动宿主的肌动蛋白细胞骨架在细胞间传播 用于细胞内运动和运输到细胞-细胞连接处。一旦到达连接点，它就会推到 膜并形成被相邻细胞吞噬的双膜突起。研究这个 独特的扩散过程将使我们能够研究几个突出的细胞生物学问题。首先， 细胞间连接处使用特定的内吞途径来吞噬微生物等大型货物？第二， 机械敏感的膜结构域或膜曲率蛋白被李斯特菌激活， 在传播过程中，？为了回答这些问题，我们使用了一种高含量的、基于图像的siRNA筛选， 测试李斯特菌在传播过程中是否需要宿主细胞间通讯途径。我们发现 内吞和机械反应性小窝蛋白CAV 1、CAV 2和PACSIN 2促进李斯特菌传播。我们 还揭示了其他19种宿主蛋白质的假定作用，包括那些调节膜曲率的蛋白质，反式- 内吞作用和粘附。我们的初步研究结果表明，李斯特菌颠覆了多重假设， 细胞间通讯途径，以促进细胞间传播。在目标1中，我们将确定PACSIN 2 和小窝蛋白协调它们的活性以促进细胞间扩散的吞噬阶段。在目标2中，我们 揭示了剩下的命中调节李斯特菌传播具体，他们如何发挥作用，如果他们的工作 独立地或与小窝一起。最后，我们提出的研究将改善我们的基本 了解宿主-微生物相互作用和基本细胞生物学，并可能揭示细胞间如何 在人类疾病中交流会出错。

PROJECT SUMMARY/ABSTRACT Multicellular organisms use intercellular communication to coordinate cell function and maintain tissue homeostasis. Recent work suggests that this communication is driven in part by the exchange of organelles (via trans-endocytosis) and mechanical cues directly across cell-cell junctions. Dysregulation of this communication leads to cancer and cardiovascular diseases. Despite its importance, we lack a fundamental molecular understanding of how intercellular communication occurs because of the limited number of cell biological tools capable of probing the molecular mechanisms at cell-cell contacts. This proposal seeks to elucidate the regulatory mechanisms of the pathways thought to control intercellular communication by studying how they are manipulated when under microbial control. The bacterium Listeria monocytogenes disseminates through human tissues using a process called cell-to-cell spread, which is a vesicular-mediated form of intercellular exchange that mimics host trans-endocytosis. Listeria spreads from cell to cell by mobilizing the host’s actin cytoskeleton for intracellular motility and transport to the cell-cell junction. Once at the junction, it pushes against the membrane and forms a double-membrane protrusion that is engulfed by a neighboring cell. Studying this distinctive spreading process will allow us to examine several outstanding cell biological questions. First, are specific endocytic pathways used at cell-cell junctions to engulf large cargo like microbes? Second, are mechanically-sensitive membrane domains or membrane curvature proteins activated as Listeria pushes against the junction during spread? To answer these questions, we used a high-content, image-based siRNA screen to test if Listeria requires host intercellular communication pathways during spread. We discovered that the endocytic and mechanoresponsive caveolar proteins CAV1, CAV2, and PACSIN2 promote Listeria spread. We also revealed a putative role for 19 other host proteins, including those that regulate membrane curvature, trans- endocytosis, and adhesion. Our preliminary findings suggest the overall hypothesis that Listeria subverts multiple intercellular communication pathways to promote cell-to-cell spread. In Aim 1, we will determine how PACSIN2 and caveolins coordinate their activities to promote the engulfment stage of cell-to-cell spread. In Aim 2, we will reveal which of the remaining hits regulate Listeria spread specifically, how they function, and if they work independently or together with caveolae. In the end, our proposed studies will improve our fundamental understanding of host-microbe interactions and basic cell biology, and may uncover how intercellular communication goes awry in human disease.